FGD5-AS1/miR-519d-3p轴调控高糖诱导的肾小管上皮细胞HK-2炎症因子表达和细胞凋亡

FGD5-AS1/miR-519d-3p axis regulates high glucose-induced inflammatory factor expression and apoptosis in renal tubular epithelial HK-2 cells

目的:探讨FGD5-AS1对高糖诱导的肾小管上皮细胞HK-2肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)表达、细胞凋亡的影响及其机制。方法:25 mmol/L葡萄糖诱导HK-2细胞24 h后,RT-qPCR检测细胞中FGD5-AS1和miR-519d-3p表达量。分别转染FGD5-AS1过表达载体(pcDNA-FGD5-AS1)、miR-519d-3p抑制剂或共转染pcDNA-FGD5-AS1与miR-519d-3p模拟物至HK-2细胞,用25 mmol/L葡萄糖诱导转染后的细胞24 h,ELISA法检测细胞培养上清液中TNF-α、IL-6水平,流式细胞术、Western blotting分别检测细胞凋亡率及凋亡相关蛋白cleaved-caspase3、cleaved-caspase9的表达。双荧光素酶报告基因实验验证FGD5-AS1和miR-519d-3p的调控关系。结果:与对照组比较,HK-2细胞经高糖诱导后,细胞中FGD5-AS1表达量明显降低(P<0.01),miR-519d-3p表达水平明显升高(P<0.01)。上调FGD5-AS1或下调miR-519d-3p的HK-2细胞经高糖诱导后,炎症因子TNF-α与IL-6水平、细胞凋亡率、凋亡相关蛋白cleaved-caspase3及cleaved-caspase9表达均降低(P<0.05~P<0.01)。FGD5-AS1靶向结合miR-519d-3p,且上调FGD5-AS1的HK-2细胞中miR-519d-3p表达明显降低(P<0.01)。过表达miR-519d-3p可逆转上调FGD5-AS1表达对高糖诱导的HK-2细胞炎症因子TNF-α与IL-6水平和细胞凋亡的影响。结论:上调FGD5-AS1可能通过靶向下调miR-519d-3p抑制高糖诱导的HK-2细胞炎症因子表达和细胞凋亡。

Objective:To investigate the effect of FGD5-AS1 on high glucose-induced tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)expression and apoptosis in renal tubular epithelial HK-2 cells and its mechanism.Methods:After HK-2 cells were induced with 25 mmol/L glucose for 24 h,the expressions of FGD5-AS1 and miR-519d-3p in the cells were detected by RT-qPCR.After HK-2 cells were transfected with FGD5-AS1 overexpression vector(pcDNA-FGD5-AS1)and miR-519d-3p inhibitor,or co-transfected with pcDNA-FGD5-AS1 and miR-519d-3p mimic,respectively,they were induced with 25 mmol/L glucose for 24 h,and then the levels of TNF-αand IL-6 in the cell culture supernatant were detected by ELISA,and the apoptosis rate and the expression of apoptosis-related proteins cleaved-caspase3 and cleaved-caspase9 were detected by flow cytometry and Western blotting,respectively.The regulatory relationship between FGD5-AS1 and miR-519d-3p was verified by the dual-luciferase reporter gene assay.Results:Compared with the control group,the expression of FGD5-AS1 in HK-2 cells induced by high glucose significantly decreased(P<0.01),and the expression of miR-519d-3p significantly increased(P<0.01).After FGD5-AS1 was up-regulated or miR-519d-3p was down-regulated in HK-2 cells,and the cells were induced by high glucose,the levels of inflammatory factors TNF-αand IL-6,apoptosis rate,expression of apoptosis-related proteins cleaved-caspase3 and cleaved-caspase9 were decreased(P<0.05 to P<0.01).FGD5-AS1 could targetedly bind to miR-519d-3p,and the expression of miR-519d-3p was significantly decreased in HK-2 cells with up-regulated FGD5-AS1(P<0.01).Overexpression of miR-519d-3p reversed the effect of up-regulating FGD5-AS1 expression on the expression of inflammatory factors TNF-αand IL-6,and apoptosis in HK-2 cells induced by high glucose.Conclusions:Up-regulation of FGD5-AS1 may inhibit high glucose-induced expression of inflammatory factors and apoptosis in HK-2 cells through targeted down-regulation of miR-519d-3p.